Search

Glucose, fructose and mannose are the preferred carbon/energy sources for the yeast Saccharomyces cerevisiae. Absence of preferred energy sources activates glucose derepression, which is regulated by the kinase Snf1. Snf1 phosphorylates the transcriptional repressor Mig1, which results in its exit from the nucleus and subsequent derepression of genes. In contrast, Snf1 is inactive when preferred carbon sources are available, which leads to dephosphorylation of Mig1 and its translocation to the nucleus where Mig1 acts as a transcription repressor. Here we revisit the role of the three hexose kinases, Hxk1, Hxk2 and Glk1, in glucose de/repression. We demonstrate that all three sugar kinases initially affect Mig1 nuclear localization upon addition of glucose, fructose and mannose. This initial import of Mig1 into the nucleus was temporary; for continuous nucleocytoplasmic shuttling of Mig1, Hxk2 is required in the presence of glucose and mannose and in the presence of fructose Hxk2 or Hxk1 is required. Our data suggest that Mig1 import following exposure to preferred energy sources is controlled via two different pathways, where (1) the initial import is regulated by signals derived from metabolism and (2) continuous shuttling is regulated by the Hxk2 and Hxk1 proteins. Mig1 nucleocytoplasmic shuttling appears to be important for the maintenance of the repressed state in which Hxk1/2 seems to play an essential role. Electronic supplementary material The online version of this article (10.1007/s00438-020-01715-4) contains supplementary material, which is available to authorized users.

Nostoc microscopicum an effective extracellular polymers (EPs) synthesizer among cyanobacteria was isolated, and its elementary morphological features were defined with the aid of light microscope and CLSM. Bioseparation of EPs from 44 days-aged culture gave 0.90 g/L of the dry powdered extract. Chemical quantification of EPs showed the presence of 550 mg/g of carbohydrate and 395 mg/g of protein. HPLC results depicted the presence of mannose (monomer sugar), fucose (hexose deoxy sugar), mannitol (sugar alcohol) and N- acetylglucosamine (glycoprotein) in the EPs. The vibration-based spectrum produced by FT-IR proves the β-Sheet structure of EPs glycoprotein and the size as 45 kDa by performing SDS-PAGE. Bactericidal activity evaluation of EPs on Pseudomonas aeruginosa and Staphylococcus aureus co-expressed the MIC value as 125 μg/mL, while zone of inhibition was 12 mm for Gram-negative and 8 mm for Gram-positive bacteria. Biofilm inhibition assay was effective in 1.0 mg/mL concentration of EPs in both bacterial strains with a mean rate of 60 percentages which was further confirmed using confocal laser scanning microscopic imaging. This natural polymeric extract of Nostoc microscopicum indicates its possible applications in bactericidal and biofilm inhibition.

International audience; The development of efficient bioprocesses requires inexpensive and renewable substrates. Molasses, a by-product of the sugar industry, contains mostly sucrose, a disaccharide composed of glucose and fructose, both easily absorbed by microorganisms. Yarrowia lipolytica, a platform for the production of various chemicals, can be engineered for sucrose utilization by heterologous invertase expression, yet the problem of preferential use of glucose over fructose remains, as fructose consumption begins only after glucose depletion what significantly extends the bioprocesses. We investigated the role of hexose transporters and hexokinase (native and fructophilic) in this preference. Analysis of growth profiles and kinetics of monosaccharide utilization has proven that the glucose preference in Y. lipolytica depends primarily on the affinity of native hexokinase for glucose. Interestingly, combined overexpression of either hexokinase with hexose transporters significantly accelerated citric acid biosynthesis and enhanced pentose phosphate pathway leading to secretion of polyols (31.5 g/L vs. no polyols in the control strain). So far, polyol biosynthesis was efficient in glycerol-containing media. Moreover, overexpression of fructophilic hexokinase in combination with hexose transporters not only shortened this process to 48 h (84 h for the medium with glycerol) but also allowed to obtain 23% more polyols (40 g/L) compared to the glycerol medium (32.5 g/L).

Cucumber (Cucumis sativus L.), an important vegetable plant species, is susceptible to low temperature stress especially during the seedling stage. Vacuolar invertase (VI) plays important roles in plant responses to abiotic stress. However, the molecular and biochemical mechanisms of VI function in cucumber, have not yet been completely understood and VI responses to low temperature stress and it functions in cold tolerance in cucumber seedlings are also in need of exploration. The present study found that hexose accumulation in the roots of cucumber seedlings under low temperature stress is closely related to the observed enhancement of invertase activity. Our genome-wide search for the vacuolar invertase (VI) genes in cucumber identified the candidate VI-encoding gene CsVI1. Expression profiling of CsVI1 showed that it was mainly expressed in the young roots of cucumber seedlings. In addition, transcriptional analysis indicated that CsVI1 expression could respond to low temperature stress. Recombinant CsVI1 proteins purified from Pichia pastoris and Nicotiana benthamiana leaves could hydrolyze sucrose into hexoses. Further, overexpression of CsVI1 in cucumber plants could increase their hexose contents and improve their low temperature tolerance. Lastly, a putative cucumber invertase inhibitor was found could form a complex with CsVI1. In summary, these results confirmed that CsVI1 functions as an acid invertase involved in hexose accumulation and responds to low temperature stress in cucumber seedlings.

Sugars, especially glucose and fructose, contribute to the taste and quality of tomato fruits. These compounds are translocated from the leaves to the fruits and then unloaded into the fruits by various sugar transporters at the plasma membrane. SWEETs, are sugar transporters that regulate sugar efflux independently of energy or pH. To date, the role of SWEETs in tomato has received very little attention. In this study, we performed functional analysis of SlSWEET7a and SlSWEET14 to gain insight into the regulation of sugar transport and storage in tomato fruits. SlSWEET7a and SlSWEET14 were mainly expressed in peduncles, vascular bundles, and seeds. Both SlSWEET7a and SlSWEET14 are plasma membrane-localized proteins that transport fructose, glucose, and sucrose. Apart from the resulting increase in mature fruit sugar content, silencing SlSWEET7a or SlSWEET14 resulted in taller plants and larger fruits (in SlSWEET7a-silenced lines). We also found that invertase activity and gene expression of some SlSWEET members increased, which was consistent with the increased availability of sucrose and hexose in the fruits. Overall, our results demonstrate that suppressing SlSWEET7a and SlSWEET14 could be a potential strategy for enhancing the sugar content of tomato fruits.

Carrageenase is useful for preparation of carrageenan oligosaccharides, which have significant bioactivity. We expressed a κ‑carrageenase gene from Zobellia sp. ZL-4 in full-length (κ-ZL-4) or after truncation of the carbohydrate binding module and the Type-IX secretion module (κ-ZL-4-GH16). κ-ZL-4-GH16 showed a specific activity (134.22 U/mg) 1.93 times higher than that of κ-ZL-4, and its thermal and pH stability also increased. The best activity of κ-ZL-4-GH16 was presented at pH 3.0–6.0, which was lower than the optimal pH of reported κ-carrageenases. The enzyme-substrate affinity of κ-ZL-4-GH16 was higher than that of κ-ZL-4, demonstrated by its lower Michaelis-Menten constant (0.704 mg/mL at pH 6.0). Importantly, κ-ZL-4-GH16 released 10-fold more κ-carrageenan disaccharides than κ-ZL-4. The κ-carrageenan tetrose and hexose produced by the two enzymes were purified and structurally identified. Molecular docking with κ-carrageenan hexose suggested that the efficiency improvement after truncation might be attributed to the conformation differences of the two enzymes.

We previously reported the precise structure of acidic free-glycans in human urine. In the present study, structural analysis of neutral free-glycans in urine was performed in fine detail. Urine samples were collected from 21 healthy volunteers and free-glycans extracted from the creatinine-adjusted urine and then fluorescently labeled with 2-aminopyridine. Neutral glycan profiling was achieved by a combination of high-performance liquid chromatography, mass spectrometry, enzymatic digestion, and periodate cleavage. A total of 79 glycans were identified. Because the ABO-blood group antigen containing urinary neutral glycans are major components, profiling patterns were similar between individuals of the same ABO-group. The neutral glycans were composed of lactose-core (Galβ1-4Glc) glycans, type-II N-acetyllactosamine-core (GlcNAcβ1-4Glc) glycans, hexose oligomers, N-glycans and to our surprise β1-3 galactosylglucose-core (Galβ1-3Glc) glycans. Although glycans with a β1-3 galactosylglucose-core were identified as major components in urine, comprising structurally simple isomers of a lactose-core, the core structure has not previously been reported. The major β1-3 galactosylglucose-core glycans were Fucα1-2Galβ1-3(Fucα1-4)Glc, GalNAcα1-3(Fucα1-2)Galβ1-3(Fucα1-4)Glc and Galα1-3(Fucα1-2)Galβ1-3(Fucα1-4)Glc, corresponding to H-, A-, and B-blood group antigens, respectively. Three lactosamine extended β1-3 galactosylglucose-core glycans were also detected as minor components. Elucidating the biosynthesis of β1-3 galactosylglucose will be crucial for understanding the in vivo function of these glycans.

The aim of this study was to evaluate the differences in the antioxidant activity and phenolic profile of sunflower (Helianthus annuus L.) extracts obtained from the aerial parts of plants harvested at five growth stages. In vitro assays were used to determine the antioxidant activity, i.e., ABTS&bull, + and DPPH&bull, scavenging activity, the ferric-reducing antioxidant power (FRAP) and the ability to inhibit &beta, carotene&ndash, linoleic acid emulsion oxidation. Phenolic compounds, such as mono- and dicaffeoylquinic acid isomers and caffeic acid hexose, were identified using the LC&ndash, TOF&ndash, MS/MS technique. The predominant compound during the growth cycle of the plant was 3,5-di-O-caffeoylquinic acid, whose content was the highest at the mid-flowering stage. The total phenolic content was also the highest in sunflowers at the mid-flowering stage. The main phenolic compound contents were closely correlated with ABTS&bull, scavenging activity and FRAP. No significant correlation was found between the total phenolic content and the antioxidant activity in the emulsion system. The highest antiradical activity and FRAP were generally determined in older plants (mid-flowering and late flowering stages). In conclusion, the aerial parts of sunflowers, in particular those harvested at the mid-flowering stage, are a good plant material from which to obtain phenolic compound extracts, albeit mainly of one class (esters of caffeic acid and quinic acid), with high antioxidant activity.

2,5-Furandicarboxylic acid (FDCA) is a promising bio-based building block as a green alternative to petroleum-based terephthalate in polymer production. Most of FDCA is produced by the oxidation of 5-hydroxymethylfurfural (HMF), which is derived from hexose. Although the chemical conversion is widely applied, the biocatalytic conversion is expected due to the relatively mild condition and fewer toxic chemicals consumption. However, it's difficult to catalyze the conversion of HMF to FDCA by a single enzyme. Here, a newly enzymatic cascade reaction process was introduced with a yield of 94.0% by the combination of 5-hydroxymethylfurfural oxidase (HMFO) and lipase. Briefly, a flavine adenosine dinucleotide independent (FAD-independent) HMFO of Methylovorus sp. MP688 was used to convert HMF to 2,5-diformylfuran (DFF) and 5-formylfuroic acid (FFA), which consecutively transformed to FDCA by a lipase Novozym 435. To facilitate the purification, a coupled alkali precipitation was developed to recover FDCA from organic solvent with an improved purity from 84.4 to 99.0% and recovery of 78.1%. This work will help to construct the green biorefinery route for the bulk FDCA from biomass by enzymes.

Evidence for the Crabtree effect was first reported by H. Crabtree in 1929 and is defined as the glucose-induced decrease of cellular respiratory flux. This effect was observed in tumor cells and was not detected in most non-tumor cells. A number of hypotheses on the mechanism underlying the Crabtree effect have been formulated. However, to this day, no consensual mechanism for this effect has been described. In a previous study on isolated mitochondria, we have proposed that fructose-1,6-bisphosphate (F1,6bP), which inhibits the respiratory chain, induces the Crabtree effect. Using whole cells from the yeast Saccharomyces cerevisiae as a model, we show here not only that F1,6bP plays a key role in the process but that glucose-6-phosphate (G6P), a hexose that has an effect opposite to that of F1,6bP on the regulation of the respiratory flux, does as well. Thus, these findings reveal that the Crabtree effect strongly depends on the ratio between these two glycolysis-derived hexose phosphates. Last, in silico modeling of the Crabtree effect illustrated the requirement of an inhibition of the respiratory flux by a coordinated variation of glucose-6-phosphate and fructose-1,6-bisphosphate to fit the respiratory rate decrease observed upon glucose addition to cells. In summary, we conclude that two glycolysis-derived hexose phosphates, G6P and F1,6bP, play a key role in the induction of the Crabtree effect.

Objective Glucose is the major energy substrate of the brain and crucial for normal brain function. In diabetes, the brain is subject to episodes of hypo- and hyperglycemia resulting in acute outcomes ranging from confusion to seizures, while chronic metabolic dysregulation puts patients at increased risk for depression and Alzheimer's disease. In the present study, we aimed to determine how glucose is metabolized in different regions of the brain using imaging mass spectrometry (IMS). Methods To examine the relative abundance of glucose and other metabolites in the brain, mouse brain sections were subjected to imaging mass spectrometry at a resolution of 100 μm. This was correlated with immunohistochemistry, qPCR, western blotting and enzyme assays of dissected brain regions to determine the relative contributions of the glycolytic and pentose phosphate pathways to regional glucose metabolism. Results In brain, there are significant regional differences in glucose metabolism, with low levels of hexose bisphosphate (a glycolytic intermediate) and high levels of the pentose phosphate pathway (PPP) enzyme glucose-6-phosphate dehydrogenase (G6PD) and PPP metabolite hexose phosphate in thalamus compared to cortex. The ratio of ATP to ADP is significantly higher in white matter tracts, such as corpus callosum, compared to less myelinated areas. While the brain is able to maintain normal ratios of hexose phosphate, hexose bisphosphate, ATP, and ADP during fasting, fasting causes a large increase in cortical and hippocampal lactate. Conclusion These data demonstrate the importance of direct measurement of metabolic intermediates to determine regional differences in brain glucose metabolism and illustrate the strength of imaging mass spectrometry for investigating the impact of changing metabolic states on brain function at a regional level with high resolution., Highlights • Utilization of glucose for glycolysis or the pentose phosphate pathway (PPP) is region-specific. • IMS allows simultaneous measurement of glucose metabolites across brain regions. • PPP is high in thalamus, while glycolysis predominates in cortex and amygdala. • Fasting induces changes in lactate distribution but not glucose metabolites or ATP.

Lactobacilli are involved in fermentation of milk-, plant-, or meat-based food products. They metabolize hexose sugars to lactic acid during fermentation, leading to a decrease in the pH. This preservative effect contributes to the digestibility of the raw material and improves the organo leptic properties of the product. Food fermentations are widely utilized in modern food production, with lactobacilli as defined starter cultures, specifically in the production of fermented raw sausages or milk products.

Adrenoleukodystrophy (X-ALD) is an X-linked genetic disorder caused by mutation of the ATP-binding cassette subfamily D member 1 gene, which encodes the peroxisomal membrane protein, adrenoleukodystrophy protein (ALDP). ALDP is associated with the transport of very-long-chain fatty acids (VLCFAs, carbon chain length ≥ 24) into peroxisomes. Defective ALDP leads to the accumulation of saturated VLCFAs in plasma and tissues, which results in damage to myelin and the adrenal glands. Here, we profiled the glycosphingolipid (GSL) species in fibroblasts from X-ALD patients. Quantitative analysis was performed using liquid chromatography–electrospray ionization–tandem mass spectrometry with a chiral column in multiple reaction monitoring (MRM) mode. MRM transitions were designed to scan for precursor ions of long-chain bases to detect GSLs, neutral loss of hexose to detect hexosylceramide (HexCer), and precursor ions of phosphorylcholine to detect sphingomyelin (SM). Our results reveal that levels of C25 and C26-containing HexCer, Hex2Cer, NeuAc-Hex2Cer, NeuAc-HexNAc-Hex2Cer, Hex3Cer, HexNAc-Hex3Cer, and SM were elevated in fibroblasts from X-ALD patients. In conclusion, we precisely quantified SM and various GSLs in fibroblasts from X-ALD patients and determined structural information of the elevated VLCFA-containing GSLs.

The mechanisms by which bacteria uptake solutes across the cell membrane broadly impact their cellular energetics. Here, we use functional genomic, genetic, and biophysical approaches to reveal how Clostridium (Lachnoclostridium) phytofermentans, a model bacterium that ferments lignocellulosic biomass, uptakes plant hexoses using highly specific, nonredundant ATP-binding cassette (ABC) transporters. We analyze the transcription patterns of its 173 annotated sugar transporter genes to find those upregulated on specific carbon sources. Inactivation of these genes reveals that individual ABC transporters are required for uptake of hexoses and hexo-oligosaccharides and that distinct ABC transporters are used for oligosaccharides versus their constituent monomers. The thermodynamics of sugar binding shows that substrate specificity of these transporters is encoded by the extracellular solute-binding subunit. As sugars are not phosphorylated during ABC transport, we identify intracellular hexokinases based on in vitro activities. These mechanisms used by Clostridia to uptake plant hexoses are key to understanding soil and intestinal microbiomes and to engineer strains for industrial transformation of lignocellulose. IMPORTANCE Plant-fermenting Clostridia are anaerobic bacteria that recycle plant matter in soil and promote human health by fermenting dietary fiber in the intestine. Clostridia degrade plant biomass using extracellular enzymes and then uptake the liberated sugars for fermentation. The main sugars in plant biomass are hexoses, and here, we identify how hexoses are taken in to the cell by the model organism Clostridium phytofermentans. We show that this bacterium uptakes hexoses using a set of highly specific, nonredundant ABC transporters. Once in the cell, the hexoses are phosphorylated by intracellular hexokinases. This study provides insight into the functioning of abundant members of soil and intestinal microbiomes and identifies gene targets to engineer strains for industrial lignocellulosic fermentation.

Colletotrichum higginsianum is an important hemibiotrophic plant pathogen that causes crucifer anthracnose worldwide. To date, some hexose transporters have been identified in fungi. However, the functions of hexose transporters in virulence are not clear in hemibiotrophic phytopathogens. In this study, we identified and characterized a new hexose transporter gene named ChHxt6 from a T-DNA insertion pathogenicity-deficient mutant G256 in C. higginsianum. Expression profiling analysis revealed that six ChHxt genes, ChHxt1 to ChHxt6, exhibited specific expression patterns in different infection phases of C. higginsianum. The ChHxt1 to ChHxt6 were separately deleted using the principle of homologous recombination. ChHxt1 to ChHxt6 deletion mutants grew normally on PDA plates, but only the virulence of ChHxt4 and ChHxt6 deletion mutants was reduced. ChHxt4 was required for fungal infection in both biotrophic and necrotrophic stages, while ChHxt6 was important for formation of necrotrophic hyphae during infection. In addition, ChHxts were functional in uptake of different hexoses, but only ChHxt6-expressing cells could grow on all five hexoses, indicating that the ChHxt6 was a central hexose transporter and crucial for hexose uptake. Site-directed mutation of T169S and P221L positions revealed that these two positions were necessary for hexose transport, whereas only the mutation Thr169 caused reduced virulence and defect in formation of necrotrophic hyphae. Taken together, ChHxt6 might regulate fungal virulence by modulating the utilization of hexose.

The metabolite profiles and antioxidant activity of Dendrobium catenatum Lindley leaf, a new functional ingredient for food product development, were evaluated in samples that had been prepared using various methods, including freeze-drying, hot-air drying, rolling before drying, steaming before drying, steaming and rolling before drying, and drying at 100, 80, and 60 °C. The concentrations of polysaccharides and flavonoids, as well as the antioxidant capacity of each sample, were determined. Furthermore, two nucleosides, four amino acids, one monoaromatic compound, and eight flavonoids were identified in dried leaves using high-performance liquid chromatography–diode array detector–electrospray ionization–multistage mass spectrometry (HPLC-DAD-ESI-MSn) and ultraviolet (UV) spectral analyses. The content of polar compounds such as cytidylic acid, arginine, tyrosine, and hydroxybenzoic acid hexose increased dramatically during hot-air-drying and rolling-before-drying treatments, while flavonol C-glycosides remained stable throughout the various treatments and drying temperatures. Rolling before drying at 100 °C was identified as the most suitable process when manufacturing tea products from D. catenatum leaves. This process resulted in a high-antioxidant-activity and visually appealing tea. This report details a potential strategy that should be applied in the manufacturing processes of high-quality products from D. catenatum leaves.

(2S,3R,4R,5S,6R)-2-Aryl-5,5-difluoro-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diols and (2S,3R,4R,5S,6R)-2-aryl-5-fluoro-5-methyl-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4-diols were discovered as dual inhibitors of sodium glucose co-transporter proteins (e.g. SGLT1 and SGLT2) through rational drug design, efficient synthesis, and in vitro and in vivo evaluation. Compound 6g demonstrated potent dual inhibitory activities (IC50 = 96 nM for SGLT1 and IC50 = 1.3 nM for SGLT2). It showed robust inhibition of blood glucose excursion in an oral glucose tolerance test (OGTT) in Sprague Dawley (SD) rats when dosed at both 1 mg/kg and 10 mg/kg orally. It also demonstrated postprandial glucose control in db/db mice when dosed orally at 10 mg/kg.

Lysine acetylation is thought to provide a mechanism for regulating metabolism in diverse bacteria. Indeed, many studies have shown that the majority of enzymes involved in central metabolism are acetylated and that acetylation can alter enzyme activity. However, the details regarding this regulatory mechanism are still unclear, specifically with regards to the signals that induce lysine acetylation. To better understand this global regulatory mechanism, we profiled changes in lysine acetylation during growth of Escherichia coli on the hexose glucose or the pentose xylose at both high and low sugar concentrations using label-free mass spectrometry. The goal was to see whether lysine acetylation differed during growth on these two different sugars. No significant differences, however, were observed. Rather, the initial sugar concentration was the principal factor governing changes in lysine acetylation, with higher sugar concentrations causing more acetylation. These results suggest that acetylation does not target specific metabolic pathways but rather simply targets accessible lysines, which may or may not alter enzyme activity. They further suggest that lysine acetylation principally results from conditions that favor accumulation of acetyl phosphate, the principal acetate donor in E. coli.IMPORTANCEBacteria alter their metabolism in response to nutrient availability, growth conditions, and environmental stresses. This process is best understood at the level of transcriptional regulation, where many metabolic genes are conditionally expressed in response to diverse cues. However, additional modes of regulations are known to exist. One is lysine acetylation, a post-translational modification known to target many metabolic enzymes. However, unlike transcriptional regulation, little is known about this regulatory mode. We investigated the factors inducing changes in lysine acetylation by comparing growth on glucose and xylose. We found that the specific sugar used for growth did not alter the pattern of acetylation; rather, the principal factor was the amount of sugar, with more sugar yielding more acetylation. These results imply lysine acetylation is a global regulatory mechanism that is not responsive to the specific carbon source per se but rather the accumulation of downstream metabolites.

Salivarian trypanosomes grow in mammals, where they depend on glucose, and as procyclic forms in tsetse flies, where they metabolize proline. Differentiation of bloodstream forms to nongrowing stumpy forms, and to procyclic forms, has been studied extensively, but reconciling the results is tricky because investigators have used parasites with various differentiation competences and different media for procyclic-form culture., Salivarian trypanosomes grow in mammals, where they depend on glucose, and as procyclic forms in tsetse flies, where they metabolize proline. Differentiation of bloodstream forms to nongrowing stumpy forms, and to procyclic forms, has been studied extensively, but reconciling the results is tricky because investigators have used parasites with various differentiation competences and different media for procyclic-form culture. Standard protocols include lowering the temperature to 27°C, adding a tricarboxylic acid, and transferring the parasites to high-proline medium, often including glucose. A 20°C cold shock enhanced efficiency. Y. Qiu, J. E. Milanes, J. A. Jones, R. E. Noorai, et al. (mSphere 3:e00366-18, 2018, https://doi.org/10.1128/mSphere.00366-18) studied this systematically, and their results call long-established protocols into question. Importantly, highly efficient differentiation was observed after cold shock and transfer to no-glucose medium without tricarboxylic acid; in contrast, glucose made differentiation tricarboxylic acid dependent and inhibited procyclic growth. New transcriptome data for stumpy and procyclic forms will enable informative comparisons with biochemical observations and with other RNA and protein data sets.

Elevated growth in breast cancer (BC) activates hypoxia-inducible factor (HIF1α) and downstream, facilitative glucose transporter 1 (GLUT1), which can be visualized with 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG). GLUT5 (fructose) and GLUT2 (glucose/fructose) might provide alternative targets for BC imaging as to why effects of hypoxia on GLUT1/2/5 levels and function were examined in human BC models. GLUT1/2/5 and HIF1α mRNA was analyzed in BC patient biopsies. In MCF10A, MCF7, and MDA-MB231 cells, [18F]FDG, 6-deoxy-6-[18F]fluoro-d-fructose (6-[18F]FDF) and [18F]-fluoroazomycin arabinoside were used in radiotracer experiments, whereas GLUT1/2/5 mRNA was analyzed with real-time PCR and protein levels determined via Western blot/immunohistochemistry. Positron emission tomography imaging was performed in MCF7 and MDA-MB231 tumor-bearing mice. Glucose/fructose/cytochalasin B reduced cellular 6-[18F]FDF uptake by 50%, indicating functional involvement of GLUT2. With GLUT5 staining lower than GLUT1, 6-[18F]FDF revealed lower uptake than [18F]FDG [standardized uptake value (SUV)6-[18F]FDF, 120 min 0.77 ± 0.06 vs. SUV[18F]FDG, 120 min 1.08 ± 0.07] in MDA-MB231 tumors and was blocked by 20% with cytochalasin B after 10 min. Whereas correspondence between 6-[18F]FDF uptake and GLUT5 protein was low, high GLUT2 levels were detected in all cell lines and tumor models. Besides GLUT1, GLUT5 seems to be regulated under hypoxia on the molecular and functional level. Additionally, results strongly support a functional involvement of GLUT2 in fructose metabolism, possibly by compensating for the weaker expression and function of GLUT5 in BC.-Hamann, I., Krys, D., Glubrecht, D., Bouvet, V., Marshall, A., Vos, L., Mackey, J. R., Wuest, M., Wuest, F. Expression and function of hexose transporters GLUT1, GLUT2, and GLUT5 in breast cancer-effects of hypoxia.

Stabilization of central carbohydrate metabolism plays a key role in plant stress response. Carbohydrates are substrate for numerous metabolic and stress-responsive reactions and have been shown to be involved in diverse signalling processes on a whole-plant level. Regulation of enzymatic sucrose synthesis and degradation is well-known to be central to many stress-related processes as it significantly impacts stress tolerance. Leaf sucrose metabolism involves sucrose cleavage by invertases and ATP-consuming resynthesis catalysed by hexokinase and sucrose phosphate synthase. These reactions establish a metabolic cycle. To study the physiological role of sucrose cycling, a kinetic model was developed to simulate dynamics of subcellular sugar concentrations in Arabidopsis thaliana under combined cold and high-light stress. Model simulation revealed that subcellular reprogramming of invertase-driven sucrose cleavage varies substantially between natural accessions of Arabidopsis which differ in their cold tolerance levels. A stress-induced shift of sucrose cleavage from the cytosol into the vacuole could only be observed for the tolerant accession while the susceptible accession increased the cytosolic proportion of sucrose cleavage. Under stress, reduction in vacuolar invertase activity significantly affected maximum quantum yield of photosystem II and CO2 assimilation rates. While wild-type plants circumvented a limitation of sucrose cleavage by increasing vacuolar invertase activity, mutant plants were not able to compensate their deficiency of vacuolar by cytosolic activity. Consequently, the capacity for cytosolic hexose generation was lower than for enzymatic hexose phosphorylation suggesting a role of vacuolar invertase activity in preventing a limitation in cytosolic hexose metabolism under stress. ENZYMES: Invertase, EC 3.2.1.26; Hexokinase, EC 2.7.1.1.

The bloodstream forms of Trypanosoma brucei (BSF), the parasite protist causing sleeping sickness, primarily proliferate in the blood of their mammalian hosts. The skin and adipose tissues were recently identified as additional major sites for parasite development. Glucose was the only carbon source known to be used by bloodstream trypanosomes to feed their central carbon metabolism, however, the metabolic behaviour of extravascular tissue-adapted parasites has not been addressed yet. Since the production of glycerol is an important primary function of adipocytes, we have adapted BSF trypanosomes to a glucose-depleted but glycerol-rich culture medium (CMM_Glyc/GlcNAc) and compared their metabolism and proteome to those of parasites grown in standard glucose-rich conditions (CMM_Glc). BSF were shown to consume 2-folds more oxygen per consumed carbon unit in CMM_Glyc/GlcNAc and were 11.5-times more sensitive to SHAM, a specific inhibitor of the plant-like alternative oxidase (TAO), which is the only mitochondrial terminal oxidase expressed in BSF. This is consistent with (i) the absolute requirement of the mitochondrial respiratory activity to convert glycerol into dihydroxyacetone phosphate, as deduced from the updated metabolic scheme and (ii) with the 1.8-fold increase of the TAO expression level compared to the presence of glucose. Proton NMR analysis of excreted end products from glycerol and glucose metabolism showed that these two carbon sources are metabolised through the same pathways, although the contributions of the acetate and succinate branches are more important in the presence of glycerol than glucose (10.2% versus 3.4% of the excreted end products, respectively). In addition, metabolomic analyses by mass spectrometry showed that, in the absence of glucose, 13C-labelled glycerol was incorporated into hexose phosphates through gluconeogenesis. As expected, RNAi-mediated down-regulation of glycerol kinase expression abolished glycerol metabolism and was lethal for BSF grown in CMM_Glyc/GlcNAc. Interestingly, BSF have adapted their metabolism to grow in CMM_Glyc/GlcNAc by concomitantly increasing their rate of glycerol consumption and decreasing that of glucose. However, the glycerol kinase activity was 7.8-fold lower in CMM_Glyc/GlcNAc, as confirmed by both western blotting and proteomic analyses. This suggests that the huge excess in glycerol kinase that is not absolutely required for glycerol metabolism, might be used for another yet undetermined non-essential function in glucose rich-conditions. Altogether, these data demonstrate that BSF trypanosomes are well-adapted to glycerol-rich conditions that could be encountered by the parasite in extravascular niches, such as the skin and adipose tissues., Author summary Until very recently, the bloodstream forms (BSF) of the Trypanosoma brucei group species have been considered to propagate exclusively in the mammalian fluids, including the blood, the lymphatic network and the cerebrospinal fluid. All these fluids are rich in glucose, which is widely considered by the scientific community as the only carbon source used by the parasite to feed its central carbon metabolism and its ATP production. Here, we show for the first time that the BSF trypanosomes efficiently grow in glucose-free conditions as long as glycerol is supplied. The raison d'être of this capacity developed by BSF trypanosomes to grow in glycerol-rich conditions regardless of the glucose concentration, including in glucose-free conditions, is not yet understood. However, the recent discovery that trypanosomes colonize and proliferate in the skin and the adipose tissues of their mammalian hosts may provide a rational explanation for the development of a glycerol-based metabolism in BSF. Indeed, the adipocytes composing adipose tissues and also abundantly present in subcutaneous layers excrete large amounts of glycerol produced from the catabolism of glucose and triglycerides. We also show that BSF trypanosomes adapted to glucose-depleted conditions activate gluconeogenesis to produce the essential hexose phosphates from glycerol metabolism. Interestingly, the constitutive expression of the key gluconeogenic enzyme fructose-1,6-bisphosphatase, which is not used for glycolysis, suggests that BSF trypanosomes maintained in the standard glucose-rich medium are pre-adapted to glucose-depleted conditions. This further strengthens the new paradigm that BSF trypanosomes can use glycerol in tissues producing this carbon source, such as the skin the adipose tissues.

We previously demonstrated that transgenic tobacco plants expressing cyanobacterial fructose-1,6-/sedoheptulose-1,7-bisphosphatase in the cytosol increased the number of lateral shoots and leaves at elevated CO2 levels. These findings suggest that alterations in carbon partitioning affect the development of shoot branching. In order to elucidate the underlying mechanisms at the molecular level, we generated transgenic Arabidopsis plants overexpressing cyanobacterial fructose-1,6-bisphosphatase-II in the cytosol (AcF). At elevated CO2 levels, the number of lateral shoots was significantly increased in AcF plants. Sucrose and hexose levels were also higher in AcF plants than in wild-type plants. The expression levels of MAX1, MAX4, YUCCA8, YUCCA9, and BRC1, which are involved in auxin or strigolactone biosynthesis and responses, were lower in AcF plants than in wild-type plants. These results suggest that alterations in sugar partitioning affect hormone metabolism and responses, resulting in enhanced shoot branching.

Most plant-pathogen interactions do not result in pathogenesis because of pre-formed defensive plant barriers or pathogen-triggered activation of effective plant immune responses. The mounting of defence reactions is accompanied by a profound modulation of plant metabolism. Common metabolic changes are the repression of photosynthesis, the increase in heterotrophic metabolism and the synthesis of secondary metabolites. This enhanced metabolic activity is accompanied by the reduced export of sucrose or enhanced import of hexoses at the site of infection, which is mediated by an induced activity of cell-wall invertase (Cw-Inv). Cw-Inv cleaves sucrose, the major transport sugar in plants, irreversibly yielding glucose and fructose, which can be taken up by plant cells via hexose transporters. These hexose sugars not only function in metabolism, but also act as signalling molecules. The picture of Cw-Inv regulation in plant-pathogen interactions has recently been broadened and is discussed in this review. An interesting emerging feature is the link between Cw-Inv and the circadian clock and new modes of Cw-Inv regulation at the post-translational level.

Two related actinomycetes, Glycomyces sp. strain NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338, were identified as potential phosphonic acid producers by screening for the gene encoding phosphoenolpyruvate (PEP) mutase, which is required for the biosynthesis of most phosphonates. Using a variety of analytical techniques, both strains were subsequently shown to produce phosphonate-containing exopolysaccharides (EPS), also known as phosphonoglycans. The phosphonoglycans were purified by sequential organic solvent extractions, methanol precipitation, and ultrafiltration. The EPS from the Glycomyces strain has a mass of 40 to 50 kDa and is composed of galactose, xylose, and five distinct partially O -methylated galactose residues. Per-deutero-methylation analysis indicated that galactosyl residues in the polysaccharide backbone are 3,4-linked Gal, 2,4-linked 3-MeGal, 2,3-linked Gal, 3,6-linked 2-MeGal, and 4,6-linked 2,3-diMeGal. The EPS from the Stackebrandtia strain is comprised of glucose, galactose, xylose, and four partially O -methylated galactose residues. Isotopic labeling indicated that the O -methyl groups in the Stackebrandtia phosphonoglycan arise from S -adenosylmethionine. The phosphonate moiety in both phosphonoglycans was shown to be 2-hydroxyethylphosphonate (2-HEP) by 31 P nuclear magnetic resonance (NMR) and mass spectrometry following strong acid hydrolysis of the purified molecules. Partial acid hydrolysis of the purified EPS from Glycomyces yielded 2-HEP in ester linkage to the O -5 or O -6 position of a hexose and a 2-HEP mono(2,3-dihydroxypropyl)ester. Partial acid hydrolysis of Stackebrandtia EPS also revealed the presence of 2-HEP mono(2,3-dihydroxypropyl)ester. Examination of the genome sequences of the two strains revealed similar pepM -containing gene clusters that are likely to be required for phosphonoglycan synthesis.

Besides the liver, which has always been considered the major source of endogenous glucose production in all post-absorptive situations, kidneys and intestines can also produce glucose in blood, particularly during fasting and under protein feeding. However, observations gained in different experimental animals have given ambiguous results concerning the presence of the glucose-6-phosphatase system in the small intestine. The aim of this study was to better define the species-related differences of this putative gluconeogenic organ in glucose homeostasis. The components of the glucose-6-phosphatase system (i.e., glucose-6-phosphate transporter and glucose-6-phosphatase itself) were analyzed in homogenates or microsomal fractions prepared from the small intestine mucosae and liver of rats, guinea pigs, and humans. Protein and mRNA levels, as well as glucose-6-phosphatase activities, were detected. The results showed that the glucose-6-phosphatase system is poorly represented in the small intestine of rats, on the other hand, significant expressions of glucose-6-phosphate transporter and of the glucose-6-phosphatase were found in the small intestine of guinea pigs and homo sapiens. The activity of the recently described fructose-6-phosphate transporter&ndash, intraluminal hexose isomerase pathway was also present in intestinal microsomes from these two species. The results demonstrate that the gluconeogenic role of the small intestine is highly species-specific and presumably dependent on feeding behavior (e.g., fructose consumption) and the actual state of metabolism.

Background: Maltose metabolism during leaf starch degradation requires a multidomain glucanotransferase and a complex polysaccharide. Results: A conventional bacterial glucanotransferase rescues an Arabidopsis mutant lacking the multidomain glucanotransferase. Conclusion: Both the plant glucanotransferase-polysaccharide couple and the bacterial enzyme provide a glucosyl buffer in the starch degradation pathway. Significance: New light is shed on the regulation and evolution of maltose metabolism., Controlled conversion of leaf starch to sucrose at night is essential for the normal growth of Arabidopsis. The conversion involves the cytosolic metabolism of maltose to hexose phosphates via an unusual, multidomain protein with 4-glucanotransferase activity, DPE2, believed to transfer glucosyl moieties to a complex heteroglycan prior to their conversion to hexose phosphate via a cytosolic phosphorylase. The significance of this complex pathway is unclear; conversion of maltose to hexose phosphate in bacteria proceeds via a more typical 4-glucanotransferase that does not require a heteroglycan acceptor. It has recently been suggested that DPE2 generates a heterogeneous series of terminal glucan chains on the heteroglycan that acts as a “glucosyl buffer” to ensure a constant rate of sucrose synthesis in the leaf at night. Alternatively, DPE2 and/or the heteroglycan may have specific properties important for their function in the plant. To distinguish between these ideas, we compared the properties of DPE2 with those of the Escherichia coli glucanotransferase MalQ. We found that MalQ cannot use the plant heteroglycan as an acceptor for glucosyl transfer. However, experimental and modeling approaches suggested that it can potentially generate a glucosyl buffer between maltose and hexose phosphate because, unlike DPE2, it can generate polydisperse malto-oligosaccharides from maltose. Consistent with this suggestion, MalQ is capable of restoring an essentially wild-type phenotype when expressed in mutant Arabidopsis plants lacking DPE2. In light of these findings, we discuss the possible evolutionary origins of the complex DPE2-heteroglycan pathway.

Recent publications suggest that high dietary fructose might play a significant role in cancer metabolism and can exacerbate a number of aspects of metabolic syndrome. Addressing the role that fructose plays in human health is a controversial question and requires a detailed understanding of many factors including the mechanism of fructose transport into healthy and diseased cells. Fructose transport into cells is thought to be largely mediated by the passive hexose transporters Glut2 and Glut5. To date, no probes that can be selectively transported by one of these enzymes but not by the other have been identified. The data presented here indicate that, in MCF-7 cells, a 1-amino-2,5-anhydro-D-mannitol-based fluorescent NBDM probe is transported twice as efficiently as fructose and that this takes place with the aid of Glut5. Its Glut5 specificity and differential uptake in cancer cells and in normal cells suggest this NBDM probe as a potentially useful tool for cross-cell-line correlation of Glut5 transport activity.

U937 cells exposed to physiological concentrations of ascorbic acid (AA) accumulate the reduced form of the vitamin in the cytosol and even further in their mitochondria. In both circumstances, uptake was dependent on Na(+) -AA-cotransport, with hardly any contribution of hexose transporters, which might be recruited to transport the oxidized form of the vitamin. There was an identical linear relationship between the mitochondrial accumulation of the vitamin and the extramitochondrial AA concentration, regardless of whether detected in experiments using intact cells or isolated mitochondria. Western blot experiments revealed expression of both SVCT1 and 2 in plasma membranes, whereas SVCT2 was the only form of the transporter expressed at appreciable amounts in mitochondria. These results therefore provide the novel demonstration of SVCT2-dependent mitochondrial transport of AA and hence challenge the present view that mitochondria only take up the oxidized form of the vitamin.

International audience; Metabolic flux analysis is particularly complex in plant cells because of highly compartmented metabolism. Analysis of free sugars is interesting because it provides data to define fluxes around hexose, pentose, and triose phosphate pools in different compartment. In this work, we present a method to analyze the isotopomer distribution of free sugars labeled with carbon 13 using a liquid chromatography ehigh resolution mass spectrometry, without derivatized procedure, adapted for Metabolic flux analysis. Our results showed a good sensitivity, reproducibility and better accuracy to determine isotopic en-richments of free sugars compared to our previous methods.

In this study, we used water-soluble stable mass isotope precursors to measure the turnover of endogenous ceramide (Cer) and glycosphingolipids (GSLs) in HEp-2 cells. Cells incubated in the presence of [13C-U]glucose showed rapid incorporation of hexose residues with an increased mass of 6Da into GSLs. Different turnover rates of GSL classes and their molecular species were observed. Approximately 30% of the glucosylceramide, 50% of the lactosylceramide, and 50% of the globotriaosylceramide species showed a much slower turnover than the rest. This demonstrates the existence of different lipid pools, where a certain fraction of species survived for a long time in the cells. The species with the shortest N-amidated fatty acyl groups (C16:0 and C18:0) showed a more rapid turnover than those with the longest N-amidated fatty acids (C24:0 and C24:1). Experiments with addition of [13C-U]serine were performed to study de novo synthesis of Cer from serine and palmitoyl-CoA. These experiments revealed that de novo synthesis contributes to a minor extent to the total synthesis of new sphingolipids and showed that there is a more rapid formation of the longest Cer species (C24:0 and C24:1) than of the shortest species (C16:0), that is, the opposite as observed for the GSLs in the experiments with [13C-U]glucose. In conclusion, this FLUX lipidomics experimental approach with the addition of [13C-U]glucose to cells allows us to not only study the total turnover but also permit observations of lipid intermediates and metabolic flow of endogenous GSL species at the molecular lipid level.

Whether nectarivores or frugivores place selective pressure on the plants they feed on, in terms of nectar or fruit traits, is much debated. Globally sugar preferences, concentration preference and digestive ability of avian nectarivores have been extensively researched. In contrast, relatively little is known about mammalian nectarivores or frugivores in terms of these, particularly Old World species. Consequently effect of sugar type and concentration on food preference in Wahlberg's epauletted fruit bat Epomophorus wahlbergi was investigated. Pair-wise choice tests were conducted using equicaloric hexose and sucrose solutions at five different concentrations (5%-25%). It was expected that they would prefer hexose sugars as these are dominant in available indigenous fruits. However, bats preferred hexoses only when offered dilute (5%) concentrations. From 10% to 25% they showed a decrease in volume intake. Their body mass was generally higher and similar after feeding during the night with the exception of 5% concentration where the mean body mass decreased. When E. wahlbergi were offered a range of sucrose or hexose solutions (10%-25%) respectively, they showed no concentration preference in terms of total volume consumed, nor energy intake. These findings suggest that these fruit bats do not appear to act as a selective pressure on sugar composition in Old World fruit. In fruit bats with high energy requirements, dietary flexibility may be an advantage when faced with seasonal and unpredictable fruit availability.

Alkaline pH stress invokes a potent and fast transcriptional response in Saccharomyces cerevisiae that includes many genes repressed by glucose. Certain mutants in the glucose-sensing and -response pathways, such as those lacking the Snf1 kinase, are sensitive to alkalinization. In the present study we show that the addition of glucose to the medium improves the growth of wild-type cells at high pH, fully abolishes the snf1 alkali-sensitive phenotype and attenuates high pH-induced Snf1 phosphorylation at Thr210. Lack of Elm1, one of the three upstream Snf1 kinases (Tos3, Elm1 and Sak1), markedly increases alkali sensitivity, whereas the phenotype of the triple mutant tos3 elm1 sak1 is even more pronounced than that of snf1 cells and is poorly rescued by glucose supplementation. DNA microarray analysis reveals that about 75% of the genes induced in the short term by high pH are also induced by glucose scarcity. Snf1 mediates, in full or in part, the activation of a significant subset (38%) of short-term alkali-induced genes, including those encoding high-affinity hexose transporters and phosphorylating enzymes. The induction of genes encoding enzymes involved in glycogen, but not trehalose, metabolism is largely dependent of the presence of Snf1. Therefore the function of Snf1 in adaptation to glucose scarcity appears crucial for alkaline pH tolerance. Incorporation of micromolar amounts of iron and copper to a glucose-supplemented medium resulted in an additive effect and allows near-normal growth at high pH, thus indicating that these three nutrients are key limiting factors for growth in an alkaline environment.

A polyhydric alcohol (PAL) was isolated from Taxus cuspidata and its immunostimulatory activities were assessed. The primary monosaccharide composition of the PAL was determined to be glucose, where HPAEC analysis showed no significant amount of any other sugars. However, glycerol and xylitol were identified as the main sugar alcohols. Fourier-transform infrared (FT-IR) analysis indicated that the purified PAL is a complex glycitol, which structurally contains significant amount of hydroxyl groups. MALDI-TOF mass spectroscopy also demonstrated that PAL is a complex glycitol built in hexose polymerization. Enzyme linked immunosorbent assay showed that the PAL stimulates the release of the proinflammatory cytokines TNF-α and IL-6 in a dose-dependent manner. Furthermore, treatment of RAW 264.7 cells with PAL for 24h remarkably increased the phosphorylation levels of ERK, p38 and JNK in a dose-dependent manner, whereas the total protein levels of ERK (t-ERK), p38 (t-p38) and JNK (t-JNK) remained unchanged. These results clearly demonstrate that PAL stimulates the immune response in RAW 264.7 cells through the activation of MAPKs (ERK, p38 and JNK) signaling pathway. To the best of our knowledge, this is the first study to demonstrate the primary structure and immune-stimulating activities of PAL from the fruit of T. cuspidata.

Glucose is required for cell growth and proliferation. The MondoA·Mlx transcription factor is glucose-responsive and accumulates in the nucleus by sensing glucose 6-phosphate. One direct and glucose-induced target of MondoA·Mlx complexes is thioredoxin-interacting protein (TXNIP). TXNIP is a potent negative regulator of glucose uptake, and hence its regulation by MondoA·Mlx triggers a feedback loop that restricts glucose uptake. This feedback loop is similar to the "hexose transport curb" first described almost 30 years ago. We show here that MondoA responds to the non-glucose hexoses, allose, 3-O-methylglucose, and glucosamine by accumulating in the nucleus and activating TXNIP transcription. The metabolic inhibitor 3-bromopyruvate blocks the transcriptional response to allose and 3-O-methylglucose, indicating that their metabolism, or a parallel pathway, is required to stimulate MondoA activity. Our dissection of the hexosamine biosynthetic pathway suggests that in addition to sensing glucose 6-phosphate, MondoA can also sense glucosamine 6-phosphate. Analysis of glucose uptake in wild-type, MondoA-null, or TXNIP-null murine embryonic fibroblasts indicates a role for the MondoA-TXNIP regulatory circuit in the hexose transport curb, although other redundant pathways also contribute.

Plant pathogenic fungi use a wide range of different strategies to gain access to the carbon sources of their host plants. The hemibiotrophic maize pathogen Colletotrichum graminicola (teleomorph Glomerella graminicola) colonizes its host plants, and, after a short biotrophic phase, switches to destructive, necrotrophic development. Here we present the identification of five hexose transporter genes from C. graminicola, CgHXT1 to CgHXT5, the functional characterization of the encoded proteins, and detailed expression studies for these genes during vegetative and pathogenic development. Whereas CgHXT4 is expressed under all conditions analyzed, transcript abundances of CgHXT1 and CgHXT3 are transiently up-regulated during the biotrophic phase, and CgHXT2 and CgHXT5 are expressed exclusively during necrotrophic development. Analyses of the encoded proteins characterized CgHXT5 as a low-affinity/high-capacity hexose transporter with a narrow substrate specificity for glucose and mannose. In contrast, CgHXT1 to CgHXT3 are high affinity/low capacity transporters that also accept other substrates, including fructose, galactose, or xylose. CgHXT4, the largest of the identified proteins, has only little transport activity and may function as a sugar sensor. Phylogenetic studies revealed hexose transporters closely related to the five CgHXT proteins also in other pathogenic fungi suggesting conserved functions of these proteins during fungal pathogenesis.

Bacillus pumilus UW-02, an isolate from agricultural soil irrigated with waste water was found to produce a carbohydrate polymer in the form of extracellular polysaccharide (EPS) in glucose mineral salts medium (GMSM). The recovery rates of EPS by ion-exchange and gel filtration chromatography were around 63% and 90%, respectively. As evident from HPLC and FT-IR analyses, the EPS was found to be a heteropolymer consisting glucose, mannose, xylose, arabinose, and N-acetyl glucosamine as monomer units. Different oligosaccharide combinations namely hexose 4 , hexose 6 pentose 1 and hexose 10 pentose 1 are obtained after partial hydrolysis of EPS using MALDI-ToF-MS. Electron micrographs portrayed the intense affinity of the EPS molecules for each other, thereby justifying its viscosifying and thickening properties. The EPS with an average molecular weight of 218 kDa and thermal stability up to 180 °C showed pseudoplastic rheology and significant emulsifying activities.

We identified and characterized two genes, LAT1 and LAT2, which encode specific L: -arabinose transporters. The genes were identified in the L: -arabinose fermenting yeast Ambrosiozyma monospora. The yeast Saccharomyces cerevisiae had only very low L: -arabinose transport activity; however, when LAT1 or LAT2 was expressed, L: -arabinose transport was facilitated. When the LAT1 or LAT2 were expressed in an S. cerevisiae mutant where the main hexose transporters were deleted, the L: -arabinose transporters could not restore growth on D: -glucose, D: -fructose, D: -mannose or D: -galactose. This indicates that these sugars are not transported and suggests that the transporters are specific for L: -arabinose.

Hexoses accumulate to high concentrations (∼ 200 mM) in storage parenchyma cells of tomato fruit. Hexoses are sourced from the fruit apoplasm as hydrolysis products of phloem-imported sucrose. Three hexose transporters (LeHT1, LeHT2, LeHT3), expressed in fruit storage parenchyma cells, may contribute to hexose uptake by these cells. An analysis of their full-length sequences demonstrated that all three transporters belong to the STP sub-family of monosaccharide transporters that localize to plasma membranes. Heterologous expression of LeHT1 (and previously LeHT2, Gear et al., 2000), but not LeHT3, rescued a hexose transport-impaired yeast mutant when raised on glucose or fructose as the sole carbon source. Biochemically, LeHT1, similarly to LeHT2, exhibited transport properties consistent with a high-affinity glucose/H(+) symporter. Significantly, LeHT1 and LeHT2 also functioned as low-affinity fructose/H(+) symporters with apparent K(m) values commensurate with those of fruit tissues. A substantial reduction (80-90%) in fruit expression levels of all LeHT genes by RNAi-mediated knockdown caused a 55% decrease in fruit hexose accumulation. In contrast, photoassimilate production by source leaves and phloem transport capacity to fruit were unaffected by transporter knockdown. Collectively, these findings demonstrate that LeHTs play key roles in driving accumulation of hexoses into storage parenchyma cells during tomato fruit development.

SUMMARYRecent research has shown that nectar properties of flowers pollinated by generalist avian nectarivores differ markedly from those of flowers pollinated by specialist avian nectarivores. In particular, flowers pollinated by generalist avian nectarivores tend to have very dilute nectar dominated by hexose sugars. To establish whether pollinator-mediated selection can explain these traits, we tested nectar sugar preferences and digestive capabilities of the village weaver (Ploceus cucullatus), a common generalist passerine nectarivore in South Africa. When offered pairwise choices of equicaloric hexose and sucrose solutions, village weavers preferred hexose solutions at 5% and 10% sucrose equivalents (SE) but did not show significant preference for either type of sugar when higher concentrations were offered (15%, 20% and 25% SE). Birds were less efficient at absorbing sucrose than hexose sugars, as revealed by high-performance liquid chromatography (HPLC) analysis of excreta sugar content. This was true at both concentrations tested (8.22% and 25%), although apparent sucrose assimilation rates were still relatively high (89.6±2.9% at low concentrations and 93.6±1.7% at high concentrations). Transit times indicated that sucrose also passes through the digestive tract faster than hexose sugars, particularly when consumed at high concentrations. This may limit the rate at which sucrose can be hydrolyzed before absorption. These results indicate that hexose preferences in generalist avian nectarivores may help explain the low sucrose content in flowers pollinated by these birds. Moreover, the preference for hexose sugars in weavers was most evident at the low concentrations (ca. 9% sugar by mass) that are typical of nectar in flowers pollinated by generalist avian nectarivores.

Summary A Plasmodium falciparum hexose transporter (PfHT) has previously been shown to be a facilitative glucose and fructose transporter. Its expression in Xenopus laevis oocytes and the use of a glucose analogue inhibitor permitted chemical validation of PfHT as a novel drug target. Following recent re-annotations of the P. falciparum genome, other putative sugar transporters have been identified. To investigate further if PfHT is the key supplier of hexose to P. falciparum and to extend studies to different stages of Plasmodium spp., we functionally analysed the hexose transporters of both the human parasite P. falciparum and the rodent parasite Plasmodium berghei using gene targeting strategies. We show here the essential function of pfht for the erythrocytic parasite growth as it was not possible to knockout pfht unless the gene was complemented by an episomal construct. Also, we show that parasites are rescued from the toxic effect of a glucose analogue inhibitor when pfht is overexpressed in these transfectants. We found that the rodent malaria parasite orthologue, P. berghei hexose transporter (PbHT) gene, was similarly refractory to knockout attempts. However, using a single cross-over transfection strategy, we generated transgenic P. berghei parasites expressing a PbHT-GFP fusion protein suggesting that locus is amenable for gene targeting. Analysis of pbht-gfp transgenic parasites showed that PbHT is constitutively expressed through all the stages in the mosquito host in addition to asexual stages. These results provide genetic support for prioritizing PfHT as a target for novel antimalarials that can inhibit glucose uptake and kill parasites, as well as unveiling the expression of this hexose transporter in mosquito stages of the parasite, where it is also likely to be critical for survival.

Rare sugars are defined as monosaccharides with extremely low natural abundances. Their natural distributions and biological functions remain to be clarified. To establish a robust analytical system that can separate, identify and quantify rare sugars, 12 d-hexoses-including five rare aldohexoses and three rare ketohexoses-were labelled with 2-aminobenzamide (2-AB), and the fluorescently tagged monosaccharides were separated by high-performance liquid chromatography (HPLC). Because the ketohexoses were much less reactive than were the aldohexoses, the reaction conditions were optimized to achieve the maximum yields (>75%) for both aldohexoses and ketohexoses. The calibration curve determined for the rare ketohexose, d-psicose (Psi), was linear between 1 pmol and 1 micromol and had a correlation coefficient of 0.9999. Using the developed method, the psicose content in a leaf of Itea virginica, in which the presence of psicose has previously been reported, was found to be 2.7 mg psicose/g leaf. The result proved feasibility of the method even for natural products. Because the system is a comprehensive tool, it should help answer questions concerning the biosyntheses and functions of rare hexose sugars.

Here we have investigated the inhibitory properties of green tea catechins on the Plasmodium falciparum hexose transporter (PfHT), the Babesia bovis hexose transporter 1 (BboHT1) and the mammalian facilitative glucose transporters, GLUT1 and GLUT5, expressed in Xenopus laevis oocytes. (-)-Epicatechin-gallate (ECG) and (-)-epigallocatechin-gallate (EGCG) inhibited D-glucose transport by GLUT1 and PfHT, and D-fructose transport by GLUT5, with apparent K(i) values between 45 and 117 microM. BboHT1 was more potently inhibited by the ungallated catechins (-)-epicatechin (EC) and (-)-epigallocatechin (EGC), with apparent K(i) values of 108 and 168 microM, respectively. Site-directed mutagenesis experiments provided little further support for previously reported models of catechin binding to hexose transporters. Furthermore, P. falciparum growth inhibition by catechins was not affected by the external D-glucose concentration. Our results provide new data on the inhibitory action of catechins against sugar transporters but were unable to elucidate the antimalarial mechanism of action of these agents.

Glucose, a ubiquitous carbon source preferred by most cells ([96][1]), was first identified by Andreas Marggraf in 1747 ([67][2]). Even though it is the most abundant hexose on earth ([33][3]), glucose is often a limiting nutrient in biological systems ([2][4]); it is a precious resource for which

Babesia are tick-transmitted haemoprotozoan parasites that infect cattle, with an estimated 500 million at risk worldwide. Here, two predicted hexose transporters (BboHT1 and 2) have been identified within the Babesia bovis genome. BboHT1, having 40% and 47% amino acid sequence similarity compared with the human (GLUT1) and Plasmodium falciparum (PfHT) hexose transporters, respectively, is the only one that could be characterised functionally after expression in Xenopus laevis oocytes. Radiotracer studies on BboHT1 showed that it is a saturable, Na(+)-independent, stereo-specific hexose transporter, with a K(m) value for glucose of 0.84+/-0.54 mM (mean+/-SEM). Using D-glucose analogues, hydroxyl positions at O-4 and O-6 have been identified as important for ligand binding to BboHT1. D-glucose transport was inhibited maximally by cytochalasin B (50 microM). A long-chain O-3 hexose derivative (compound 3361) that selectively inhibits PfHT also inhibited relatively potently BboHT1, with an apparent K(i) value of 4.1+/-0.9 microM (mean+/-SEM). Compound 3361 did not inhibit B. bovis proliferation in in vitro growth assays but inhibited invasion of glucose-depleted bovine erythrocytes. Taken together with results of inhibition studies with cytochalasin B and beta-glucogallin, these data provide new insights into glucose metabolism of erythrocytic-stage Babesia infections.

In numerous cell types, tumoral cells, proliferating cells, bacteria, and yeast, respiration is inhibited when high concentrations of glucose are added to the culture medium. This phenomenon has been named the "Crabtree effect." We used yeast to investigate (i) the short term event(s) associated with the Crabtree effect and (ii) a putative role of hexose phosphates in the inhibition of respiration. Indeed, yeast divide into "Crabtree-positive," where the Crabtree effect occurs, and "Crabtree-negative," where it does not. In mitochondria isolated from these two categories of yeast, we found that low, physiological concentrations of glucose 6-phosphate and fructose 6-phosphate slightly (20%) stimulated the respiratory flux and that this effect was strongly antagonized by fructose 1,6-bisphosphate (F16bP). On the other hand, F16bP by itself was able to inhibit mitochondrial respiration only in mitochondria isolated from a Crabtree-positive strain. Using permeabilized spheroplasts from Crabtree-positive yeast, we have shown that the sole effect observed at physiological concentrations of hexose phosphates is an inhibition of oxidative phosphorylation by F16bP. This F16bP-mediated inhibition was also observed in isolated rat liver mitochondria, extending this process to mammalian cells. From these results and taking into account that F16bP is able to accumulate in the cell cytoplasm, we propose that F16bP regulates oxidative phosphorylation and thus participates in the establishment of the Crabtree effect.

A novel beta-galactosidase of 120 kDa (BgaBM) from Bacillus megaterium 2-37-4-1 was purified, and its gene (bgaBM) was analyzed and expressed. It displayed wide acceptor specificity for transglycosylation with a series of acceptors, including pentose, hexose, hydroxyl, and alkyl alcohol using o-nitrophenyl-beta-D-galactoside (ONPG) as a donor. BgaBM preferentially hydrolyzed ONPG in all tested substrates and showed maximum activity at pH 7.5-8.0 and 55 degrees C. It was stable at pH 6.0-9.0 below 40 degrees C. The K(m) and V(max) values for ONPG and lactose were 9.5 mM, 16.6 mM/min and 12.6 mM, 54.4 mM/min, respectively. The nucleotide sequence of the bgaBM gene consists of an ORF of 3,105 bp corresponding to 118 kDa protein, which indicates that BgaBM is a modular enzyme in the glycosyl hydrolase family 2, including conserved sugar-binding domain, acid-base catalyst, and immunoglobulin-like beta-sandwich domain. The possible acid/base and nucleophile sites of BgaBM were estimated to be E481 and E547, respectively. Furthermore, expression of the bgaBM gene in Escherichia coli and purification of the recombinant enzyme were performed. The recombinant enzyme showed similar biochemical characteristics to natural enzyme.